Comparative vaccine efficacy of different isoforms of recombinant protective antigen against Bacillus anthracis spore challenge in rabbits.

The next-generation human anthrax vaccine developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) is based upon purified Bacillus anthracis recombinant protective antigen (rPA) adsorbed to aluminum hydroxide adjuvant (Alhydrogel). In addition to being safe, and effective, it is important that such a vaccine be fully characterized. Four major protein isoforms detected in purified rPA by native PAGE during research and development were reduced to two primary isoforms in bulk material produced by an improved process performed under Good Manufacturing Practices (GMP). Analysis of both rPA preparations by a protein-isoaspartyl-methyl-transferase assay (PIMT) revealed the presence of increasing amounts of iso-aspartic acid correlating with isoform content and suggesting deamidation as the source of rPA charge heterogeneity. Additional purification of GMP rPA by anion exchange chromatography separated and enriched the two principal isoforms. The in vitro and in vivo biological activities of each isoform were measured in comparison to the whole GMP preparation. There was no significant difference in the biological activity of each isoform compared to GMP rPA when analyzed in the presence of lethal factor using a macrophage lysis assay. Vaccination with the two individual isoforms revealed no differences in cytotoxicity neutralization antibody titers when compared to the GMP preparation although one isoform induced more anti-PA IgG antibody than the GMP material. Most importantly, each of the two isoforms as well as the whole GMP preparation protected 90-100% of rabbits challenged parenterally with 129 LD50 of B. anthracis Ames spores. The equivalent biological activity and vaccine efficacy of the two isoforms suggests that further processing to separate isoforms is unnecessary for continued testing of this next-generation anthrax vaccine.

Chromosomal DNA deletions explain phenotypic characteristics of two antigenic variants, phase II and RSA 514 (crazy), of the Coxiella burnetii nine mile strain.

After repeated passages through embyronated eggs, the Nine Mile strain of Coxiella burnetii exhibits antigenic variation, a loss of virulence characteristics, and transition to a truncated lipopolysaccharide (LPS) structure. In two independently derived strains, Nine Mile phase II and RSA 514, these phenotypic changes were accompanied by a large chromosomal deletion (M. H. Vodkin and J. C. Williams, J. Gen. Microbiol. 132:2587-2594, 1986). In the work reported here, additional screening of a cosmid bank prepared from the wild-type strain was used to map the deletion termini of both mutant strains and to accumulate all the segments of DNA that comprise the two deletions. The corresponding DNAs were then sequenced and annotated. The Nine Mile phase II deletion was completely nested within the deletion of the RSA 514 strain. Basic alignment and homology studies indicated that a large group of LPS biosynthetic genes, arranged in an apparent O-antigen cluster, was deleted in both variants. Database homologies identified, in particular, mannose pathway genes and genes encoding sugar methylases and nucleotide sugar epimerase-dehydratase proteins. Candidate genes for addition of sugar units to the core oligosaccharide for synthesis of the rare sugar 6-deoxy-3-C-methylgulose (virenose) were identified in the deleted region. Repeats, redundancies, paralogous genes, and two regions with reduced G+C contents were found within the deletions.

A Francisella tularensis DNA clone complements Escherichia coli defective for the production of Era, an essential Ras-like GTP-binding protein.

We cloned the era gene of Francisella tularensis from a plasmid library by heterologous genetic complementation of an Escherichia coli mutant conditionally defective for the production of Era, an essential protein for cell growth. Nucleotide sequence analysis indicated that, in F. tularensis, era constitutes a single gene operon. ORFs aspC and mdh encoding aspartate aminotransferase and malate dehydrogenase, respectively, flank era in F. tularensis. Although classified as Gram-, the flanking regions and the relative location of era in F. tularensis are distinctly different from those of typical Gram- and Gram+ bacteria. Computer analysis of bacterial Era protein sequences identified conserved domains in addition to the common G domains of most GTP-binding proteins.

Analysis of the DnaK molecular chaperone system of Francisella tularensis.

We have cloned the Francisella tularensis (Ft) grpE-dnaK-dnaJ heat-shock genes which are organized in that order. These genes allow heterologous genetic complementation of each respective mutant strain of Escherichia coli (Ec) for bacteriophage lambda growth. The nucleotide sequences of the Ft grpE-dnaK-dnaJ genes and the deduced amino-acid sequences share significant homologies with their respective Ec counterparts. The Ft DnaK and DnaJ proteins cross-react with polyclonal antibodies raised against the respective Ec proteins. The grpE-dnaK-dnaJ genes of Ft are organized in a fashion that is more characteristic of Gram+ bacteria.

Analysis of a Coxiella burnetti gene product that activates capsule synthesis in Escherichia coli: requirement for the heat shock chaperone DnaK and the two-component regulator RcsC.

A 1.2-kb EcoRI genomic DNA fragment of Coxiella burnetti, when cloned onto a multicopy plasmid, was found to induce capsule synthesis (mucoidy) in Escherichia coli. Nucleotide sequence analysis revealed the presence of an open reading frame that could encode a protein of 270 amino acids. Insertion of a tet cassette into a unique NruI restriction site resulted in the loss of induction of mucoidy. Because of its ability to induce mucoidy, we designated this gene mucZ. Computer search for homologies to mucZ revealed 42% identity to an open reading frame located at 1 min of the E. coli chromosome. Interestingly, the C-terminal amino acid residues of MucZ share significant homology with the J domain of the DnaJ protein and its homologs, suggesting potential interactions between MucZ and components of the DnaK-chaperone machinery. Results presented in this paper suggest that E. coli requires DnaK-chaperone machinery for Lon-RcsA-mediated induction of capsule synthesis, as noticed first by S. Gottesman (personal communication). The induction caused by MucZ is independent of Lon-RcsA and is mediated through the two-component regulators RcsC and RcsB. DnaK and GrpE but not DnaJ are also required for the RcsB-mediated MucZ induction, and we propose that MucZ is a DnaJ-like chaperone protein that might be required for the formation of an active RcsA-RcsB complex and for the RcsC-dependent phosphorylation of RcsB. Discussions are presented that suggest three different roles for alternative forms of the DnaK-chaperone machinery in capsule production.

Identification and characterization of a trans-activator involved in the regulation of encapsulation by Bacillus anthracis.

Production of the plasmid-pXO2-encoded capsule by Bacillus anthracis is required for full virulence of the organism. The induction of capsule synthesis in vitro requires growth in the presence of bicarbonate and CO2; however, little else is known about the regulation of capsule synthesis and the role it plays in the expression of virulence. Recently, transposon Tn917 mutagenesis of B. anthracis plasmid pXO2 identified genes involved in capsule production and genes associated with virulence in inbred mice. One mutant, UUP5, had an 8.2-kb deletion located outside of the capsule structural gene region (cap). UUP5 was reduced significantly in capsule production and in virulence as compared to the wild-type (wt) parental strain. Using a HindIII-generated pXO2 library, we examined fragments contained in the deleted region and showed that electroporation of the mutant with a cloned 2.3-kb HindIII fragment restored capsule production to wt levels. Sequence analysis of the 2.3-kb fragment revealed a 1449-bp open reading frame (ORF) encoding a 483-amino-acid (57 kDa) protein, in good agreement with the 55-kDa protein detected by in vitro transcription/translation. Construction of a frameshift mutant that replaced the 55-kDa protein with a truncated 34-kDa moiety abrogated the complementing activity of the fragment in UUP5. mRNAs specific for cap and for the 1449-bp ORF were detected in mutant UUP5 transformed with the unaltered fragment and grown in the presence of bicarbonate, but not in air. No cap-specific mRNA, and very low levels of ORF-specific mRNA, were detected in UUP5 containing the frameshift mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, sequencing and expression of the dnaJ gene of Coxiella burnetii.

A 6-kb EcoRI genomic DNA fragment of Coxiella burnetii, isolated from a recombinant bacteriophage lambda ZapII library, allowed heterologous genetic complementation of Escherichia coli deleted for its dnaJ gene. The C. burnetii dnaJ gene was expressed in E. coli and identified by Western blot analysis using polyclonal antibodies raised against purified E. coli DnaJ protein. Deletion mapping and genetic complementation demonstrated that C. burnetii dnaJ is present on a 2-kb EcoRI-HindIII genomic DNA fragment, from which the nt sequence of the C. burnetii dnaJ gene was determined.

Analysis of the rnc locus of Coxiella burnetii.

A 3.2 kb EcoRI genomic DNA fragment of Coxiella burnetii was isolated by virtue of its ability to suppress mucoidy in Escherichia coli. Nucleotide sequence analysis revealed the presence of the genes homologous to rnc, era and recO of E. coli. Suppression of capsule synthesis, measured by beta-galactosidase expression in lon- cps-lac fusion strains of E. coli, is caused by gene-dosage effects of the plasmid-borne rnc genes of either C. burnetii or E. coli. The rnc gene of C. burnetii complemented rnc- E. coli hosts for lambda plaque morphology and stimulation of lambda N gene expression. We also demonstrated heterologous complementation of an E. coli strain defective for the expression of Era, an essential protein in E. coli, using the plasmid-borne C. burnetii era. Under the control of the bacteriophage lambda PL promoter, this 3.2 kb EcoRI DNA fragment directed the synthesis in E. coli of three proteins with approximate molecular masses of 35, 27 and 25 kDa. Antibodies against purified E. coli Era protein cross-reacted with the 35 kDa protein of C. burnetii on Western blots.

Cloning and characterization of an autonomous replication sequence from Coxiella burnetii.

A Coxiella burnetii chromosomal fragment capable of functioning as an origin for the replication of a kanamycin resistance (Kanr) plasmid was isolated by use of origin search methods utilizing an Escherichia coli host. The 5.8-kb fragment was subcloned into phagemid vectors and was deleted progressively by an exonuclease III-S1 technique. Plasmids containing progressively shorter DNA fragments were then tested for their capability to support replication by transformation of an E. coli polA strain. A minimal autonomous replication sequence (ARS) was delimited to 403 bp. Sequencing of the entire 5.8-kb region revealed that the minimal ARS contained two consensus DnaA boxes, three A + T-rich 21-mers, a transcriptional promoter leading rightwards, and potential integration host factor and factor of inversion stimulation binding sites. Database comparisons of deduced amino acid sequences revealed that open reading frames located around the ARS were homologous to genes often, but not always, found near bacterial chromosomal origins; these included identities with rpmH and rnpA in E. coli and identities with the 9K protein and 60K membrane protein in E. coli and Pseudomonas species. These and direct hybridization data suggested that the ARS was chromosomal and not associated with the resident plasmid QpH1. Two-dimensional agarose gel electrophoresis did not reveal the presence of initiating intermediates, indicating that the ARS did not initiate chromosome replication during laboratory growth of C. burnetii.

A Coxiella burnetti repeated DNA element resembling a bacterial insertion sequence.

A DNA fragment located on the 3' side of the Coxiella burnetii htpAB operon was determined by Southern blotting to exist in approximately 19 copies in the Nine Mile I genome. The DNA sequences of this htpAB-associated repetitive element and two other independent copies were analyzed to determine the size and nature of the element. The three copies of the element were 1,450, 1,452, and 1,458 bp long, with less than 2% divergence among the three sequences. Several features characteristic of bacterial insertion sequences were discovered. These included a single significant open reading frame that would encode a 367-amino-acid polypeptide which was predicted to be highly basic, to have a DNA-binding helix-turn-helix motif, to have a leucine zipper motif, and to have homology to polypeptides found in several other bacterial insertion sequences. Identical 7-bp inverted repeats were found at the ends of all three copies of the element. However, duplications generated by many bacterial mobile elements in the recipient DNA during insertion events did not flank the inverted repeats of any of the three C. burnetii elements examined. A second pair of inverted repeats that flanked the open reading frame was also found in all three copies of the element. Most of the divergence among the three copies of the element occurred in the region between the two inverted repeat sequences in the 3' end of the element. Despite the sequence changes, all three copies of the element have retained significant dyad symmetry in this region.

Antigenic structure of Coxiella burnetii. A comparison of lipopolysaccharide and protein antigens as vaccines against Q fever.

The antigenic structure of Coxiella burnetii is being investigated by identifying both external and internal cellular epitopes of the morphologic cell types. Both the phase I lipopolysaccharide (LPSI) and several surface proteins are candidates for the development of subunit multivalent vaccines. The protective efficacy of purified LPSI was demonstrated in A/J mice. The purified LPSI preparations contained residual peptides detected by amino acid analysis. Therefore, the protection afforded by LPSI may be, in part, due to the presence of peptides. The purification of proteins free of LPSI must be accomplished before the protective efficacy of proteins or peptides can be established. We have identified three proteins that are both antigenic and immunogenic, as indicated by either enzyme immunoassay, radioimmunoprecipitation, immunoblot assay, or lymphocyte transformation. A 62-kDa protein antigen encoded by the htpB gene of C. burnetii was analyzed for immunogenicity. The purified protein antigen was immunogenic, as it elicited specific antibodies and performed as recall antigen in lymphocyte stimulation assays. The antigen was not detected on the surface of phase I cells but was highly represented on the surface of phase II cells. Therefore, the protein may not be a good candidate for vaccine development. The diagnostic utility of the 62-kDa protein antigen lies in the fact that convalescent and chronic Q fever sera from human patients reacted with the antigen, whereas acute sera did not. Although the 62-kDa protein is a "common antigen," specific peptide-based diagnostic reagents may be useful in the detection of Q fever disease progression. A major surface protein (P1) of roughly 29.5 kDa was purified from the phase I Nine Mile (clone 7) strain. No LPSI was detected in the P1 preparation by three different LPSI monoclonal antibodies. Monoclonal antibodies prepared against P1 were effective in localizing the protein on the cell surface, in the cell wall, and associated with the peptidoglycan of large cells of C. burnetii. Small, pressure-resistant cells did not contain P1. Mice immunized with two 25-micrograms injections of LPSI produced antibodies against LPSI and phase I whole cells. No antibody was detected against phase II whole cells. Immunization with P1 induced antibody against the LPSI fraction and phase I and phase II whole cells. P1 was more effective than LPSI in reducing the number of infectious C. burnetii in the spleens of challenged mice. The gene encoding another protein (P2) recognized by P1 monoclonal antibodies was cloned and sequenced.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of Coxiella burnetii pyrB.

The C. burnetii pyrB gene was cloned on a 7-kbp EcoR I fragment. DNA sequence analysis, enzyme assays, and amino acid homologies with E. coli and B. subtilis pyrB gene products suggest that (i) C. burnetii ATCase exists as a trimer, (ii) the microorganism may not synthesize a regulatory polypeptide, and (iii) pyrB may be part of an operon whose expression is under the control of an upstream promoter. The high degree of homology of the active site further suggests that a common mechanism of catalysis for ATCase exists between such diverse organisms as C. burnetii, E. coli, and B. subtilis.

Site-specific substitutions of the Tyr-165 residue in the catalytic chain of aspartate transcarbamoylase promotes a T-state preference in the holoenzyme.

The amino acid residue Tyr-165C of aspartate transcarbamoylase (EC 2.1.3.2) of Escherichia coli has been proposed to be involved in the transition from the T-state to the R-state upon binding of the bisubstrate analogue N-(phosphonacetyl)-L-aspartate. Site-specific mutagenesis has been used to substitute phenylalanine for tyrosine, thus maintaining the aromatic R-group but removing the charged hydroxyl moiety. This mutation dramatically altered the aspartate requirements for the holoenzyme but did not substantially affect the homotropic or heterotropic characteristics of the oligomer. The aspartate requirements for half-maximal saturation increased from 5.5 mM at pH 7.0 for the native holoenzyme to approximately 90 mM in the mutant enzyme. Nonetheless, estimates of the kinetic cooperativity index remained similar (Hill coefficients: Tyr-165C, n = 2.1; Phe-165C, n = 2.5). CTP inhibited both enzymes approximately 70% and ATP activated approximately 40% at the aspartate concentrations required for half-maximal saturation (5 and 90 mM, respectively). The maximal velocity of the mutant holoenzyme is almost identical to that of the wild-type enzyme. The phenylalanine substitution does not affect the stability of the holoenzyme to heat or mercurials, and the Vmax of the catalytic trimer was 444% greater than that of the holoenzyme. Upon dissociation of the wild-type native enzyme into catalytic trimers, the Vmax increased 450%. The Km for aspartate in the separated catalytic trimer is approximately 2-fold higher than for the native catalytic trimer (16.5 versus 8 mM at pH 7.0). It is clear from the data that although Tyr-165C is not directly involved in the active site of the enzyme, it does play a pivotal role in catalytic transitions of the holoenzyme. In addition, the homotropic and heterotropic characteristics of the enzyme do not seem to be altered by the substitution of phenylalanine for Tyr-165C in the E. coli aspartate transcarbamoylase, although other substitutions have been reported (Robey, E. H., and Schachman, H. K. (1984) J. Biol. Chem. 259, 11180-11183) which show more complex effects.

The DNA sequence of argI from Escherichia coli K12.

The argI gene from E. coli K12 has been sequenced. It contains an open reading frame of 1002 bases which encodes a polypeptide of 334 amino acids. Three such polypeptides are required to form the functional catalytic trimer (c3) of ornithine transcarbamoylase (OTCase-1, EC 2.1.3.3). The molecular mass of the mature trimer deduced from the amino acid sequence is 114,465 daltons. An altered form of argI was produced when a 1.6 kilobase DdeI fragment was subcloned into the HincII site of plasmid pUC8 extending the open reading frame an additional 20 nucleotides. It has been previously reported that the amino-terminal region of the respective polypeptides of argI, argF, and pyrB of E. coli possessed significant homology. In contrast, the homologous promoter/operator regions of argI and argF did not appear to share any homologies with pyrB. However, a closer scrutiny of the nucleotide sequence immediately preceding the pyrBI attenuator revealed a remarkable similarity to the argI and argF control region.

Nucleotide sequence of the structural gene (pyrB) that encodes the catalytic polypeptide of aspartate transcarbamoylase of Escherichia coli.

The deoxyribonucleotide sequence of pyrB, the cistron encoding the catalytic subunit of aspartate transcarbamoylase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2), has been determined. The pyrB gene encodes a polypeptide of 311 amino acid residues initiated by an NH2-terminal methionine that is not present in the catalytically active polypeptide. The DNA sequence analysis revealed the presence of an eight-amino-acid sequence beginning at Met-219 that was not detected in previous analyses of amino acid sequence. This octapeptide sequence provides an additional component of the disordered loop in the equatorial domain of the catalytic polypeptide. It had been found previously that the catalytic polypeptide is expressed from a bicistronic operon that also produces the regulatory polypeptide encoded by pyrI. A single transcriptional control region precedes the structural gene of the catalytic polypeptide and a simple 15-base-pair region separates its COOH terminus from the structural gene of the regulatory polypeptide. The chain-terminating codon of the catalytic polypeptide may contribute to the ribosomal binding site for the regulatory polypeptide and thus assist coordinate expression of the two cistrons.